Both hematopoietic failure and development of extramedullary hematopoiesis are associated with disease progression in patients with primary myelofibrosis (PMF). During the previous funding period, close cooperation between Project 4 and 5 led to the identification of striking similarities between stem/progenitor cell (HSC/HPC) and microenvironmental (HM) abnormalities present in PMF patients and Gatallow mice, an animal model for this disease. These abnormalities are potential targets for drug development and we have already identified plitidepsin as a drug that altered the natural history of myelofibrosis in Gatallow mice by targeting both abnormalities. This drug is presently under investigation in Project 6 for the treatment of PMF patients (MPD-RC 110). In this project, we plan to continue the fruitful interaction between Project 4 and 5 by further defining HSC/HPC and HM abnormalities associated with myelofibrosis in Gatallow mice. By transplantation assay and forced gene expression, we propose to test the hypothesis that the hematopoietic failure and development of extramedullary hematopoiesis in Gatallow mice with myelofibrosis is due to autonomous defects of HSC/HPC due to insufficient expression of CXCR4 and/or Rad (respectively the receptor and the first intracellular signaling molecule of the chemokine CXCR12 (Specific aim 1). By immuno-electron microscopy studies and loss of function experiments, we propose to test the hypothesis that, because of alterations in protein sorting into the D-granules, Gatallow MK (and possibly MK from PMF patients) stimulate mesenchymal stem cell maturation into osteoblasts impairing the ability of these cells to form a functional HM in the marrow (Specific aim 2). In addition, in vivo treatments of Gatallow mice with inhibitors of JAK2, the neutrophil protease MMP-9 and TGFD will test the hypothesis that drugs targetting abnormalities in HSC/HPC and HM of Gatal low mice will improve the natural history of myelofibrosis, alone or in combination with plitidepsin, in this animal model of the disease (Specific aim 3),